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Merging pathology with biomechanics using CHIMERA (Closed-Head Impact Model of Engineered Rotational… Namjoshi, Dhananjay R; Cheng, Wai H; McInnes, Kurt A; Martens, Kris M; Carr, Michael; Wilkinson, Anna; Fan, Jianjia; Robert, Jerome; Hayat, Arooj; Cripton, Peter A; Wellington, Cheryl L Dec 1, 2014

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RESEARCH ARTICLE Open AccessMerging pathology with biomechanics usingCHIMERA (Closed-Head ImConclusions: Repeated TBI using CHIMERA mimics many of the functional and pathological characteristics of humanNamjoshi et al. Molecular Neurodegeneration 2014, 9:55, Vancouver, BC, CanadaFull list of author information is available at the end of the articleTBI with a reliable biomechanical response of the head. This makes CHIMERA well suited to investigate thepathophysiology of TBI and for drug development programs.Keywords: Traumatic brain injury, Animal model of traumatic brain injury, Animal model of closed head injury, Diffuseaxonal injury, Microglia activation, Neuroinflammation, Tau hyperphosphorylation, Head kinematics, Head injurybiomechanics, Impact-acceleration traumatic brain injury model, Surgery-free animal model of traumatic brain injury,Traumatic brain injury biomechanics* Correspondence:†Equal contributors1Department of Pathology and Laboratory Medicine, The University of BritishColumbia, Vancouver, BC, Canada4International Collaboration on Repair Discoveries, The University of BritishEngineered Rotational Acceleration): a novel,surgery-free model of traumatic brain injuryDhananjay R Namjoshi1,2†, Wai Hang Cheng1†, Kurt A McInnes3,4, Kris M Martens1, Michael Carr1, Anna Wilkinson1,Jianjia Fan1, Jerome Robert1, Arooj Hayat1, Peter A Cripton3,4 and Cheryl L Wellington1,4,5*AbstractBackground: Traumatic brain injury (TBI) is a major health care concern that currently lacks any effective treatment.Despite promising outcomes from many preclinical studies, clinical evaluations have failed to identify effectivepharmacological therapies, suggesting that the translational potential of preclinical models may require improvement.Rodents continue to be the most widely used species for preclinical TBI research. As most human TBIs result fromimpact to an intact skull, closed head injury (CHI) models are highly relevant, however, traditional CHI models sufferfrom extensive experimental variability that may be due to poor control over biomechanical inputs. Here we describe anovel CHI model called CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) that fullyintegrates biomechanical, behavioral, and neuropathological analyses. CHIMERA is distinct from existing neurotraumamodel systems in that it uses a completely non-surgical procedure to precisely deliver impacts of prescribed dynamiccharacteristics to a closed skull while enabling kinematic analysis of unconstrained head movement. In this study, wecharacterized head kinematics as well as functional, neuropathological, and biochemical outcomes up to 14d followingrepeated TBI (rTBI) in adult C57BL/6 mice using CHIMERA.Results: Head kinematic analysis showed excellent repeatability over two closed head impacts separated at 24h.Injured mice showed significantly prolonged loss of righting reflex and displayed neurological, motor, and cognitivedeficits along with anxiety-like behavior. Repeated TBI led to diffuse axonal injury with extensive microgliosis in whitematter from 2-14d post-rTBI. Injured mouse brains also showed significantly increased levels of TNF-α and IL-1β andincreased endogenous tau phosphorylation.© 2014 Namjoshi et al.; licensee BioMed CentCommons Attribution License (http://creativecreproduction in any medium, provided the orDedication waiver (http://creativecommons.orunless otherwise stated.pact Model ofral Ltd. This is an Open Access article distributed under the terms of the, which permits unrestricted use, distribution, andiginal work is properly credited. The Creative Commons Public Domaing/publicdomain/zero/1.0/) applies to the data made available in this article,Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 2 of 18 brain injury (TBI) is a leading worldwide causeof death and disability for persons under 45 years of agewith a cost to society of over 17 billion USD per year. Inthe United States, the overall incidence of TBI is estimatedto be 538 per 100,000 persons, which represents at least1.7 million new cases per year since 2003 [1-3]. TBIincidence is reportedly lower in Europe (235 per 100,000)and Australia (322 per 100,000) [4,5] although recentepidemiological data suggests far greater incidence (749per 100,000) [6]. Mild TBI (mTBI), which includes con-cussion, comprises over 75% of all TBIs [3]. As mTBI isincreasingly recognized as an injury for which medicalattention should be sought, the reported incidence ofmTBI is rising.Falls are the most prevalent cause of TBI, and motorvehicle accidents and impacts against objects are alsocommon causes [2,4,7]. TBI resulting from high-contactsports such as boxing, American football, ice hockey,soccer, and rugby account for almost 21% of all head in-juries among children and adolescents, particularly formTBI [8]. In these situations, the skull experiences animpact resulting in brain deformation and resulting in-jury that most often occurs without skull fracture. TBI isalso considered a “signature injury” in modern warfare,as approximately 20% of veterans from the Iraq orAfghanistan wars are reported to have experienced aTBI, 80% of which involve both blunt impact and over-pressure mechanisms [9-12]. Furthermore, the growingawareness that mTBI may have long-lasting and severeconsequences [13-16] highlights the urgency to under-stand much more about the acute and long-term conse-quences of brain injury.Rapid acceleration and rotation during impact TBI leadto vigorous movement and deformation of brain tissuewithin the skull that can result in contusions as the braincontacts the interior of the bony skull. These inertial andcontact forces directly affect neurons, blood vessels, andglia, producing a primary injury that initiates secondaryprocesses within hours to weeks after the initial injury[17-22]. These secondary changes lead to a plethora ofevents including edema, raised intracranial pressure, im-paired cerebral blood flow, increased blood–brain barrierpermeability, inflammation, axonal injury, calcium influx,elevated oxidative stress, free radical-mediated damage,excitatory neurotransmitter release, and cell death [17-22].Although few treatment options are available for theprimary injury, secondary injury pathways are potentiallymodifiable [23]. An increasingly wide variety of experi-mental animal models are therefore being developed tocharacterize secondary injury processes and for the evalu-ation of candidate therapeutic approaches.No single animal model can replicate the entire spectrumof human TBI pathophysiology; therefore, several large andsmall animal models have been developed to mimic par-ticular aspects. Popular rodent models include open-headinjury models such us fluid percussion (FP) and controlledcortical impact (CCI) systems, and closed-head injury(CHI) models that use either gravity or mechanicalmethods to impact the intact skull (reviewed in [24]). Al-though FP and CCI models employ highly reproduciblemechanical inputs and can mimic many pathological fea-tures of human TBI, the prominent tissue destruction andlack of head movement in these models decreases their re-semblance to the majority of human injuries that occur dueto impact and/or acceleration on an intact skull. In con-trast, closed-head injury (CHI) models employ methodsthat do not generally cause overt brain tissue loss and canalso allow rapid behavioral assessment of injury severity. Assuch, CHI models are considered by some to better mimicthe majority of human TBI cases. However, a major limi-tation of most current CHI animal models is that theinput parameters used to induce injury (e.g., mechanicalloading, method of mechanical input, and response of theanimal’s head to mechanical loading) are often poorlycontrolled, which can contribute to the considerableexperimental variation across cognitive, histological, andbiochemical outcome measures (reviewed in [24]).Here we report a novel neurotrauma model calledCHIMERA (Closed-Head Impact Model of EngineeredRotational Acceleration). CHIMERA was developed toaddress the absence of a simple and reliable model of ro-dent CHI that is representative of the majority of humanTBI cases. CHIMERA is distinct from existing neuro-trauma model systems in that it fully integrates biomech-anical, behavioral, and neuropathological analyses afterdelivering impacts of defined energy to a closed skull withunconstrained head motion after impact. Here we showthat repeated TBI (rTBI) in mice using CHIMERA reliablyinduces motor deficits, anxiety-like behavior, memoryimpairment, and leads to persistent diffuse axonal injury(DAI) with extensive white matter inflammation andincreased phosphorylation of endogenous tau.ResultsHead kinematics during CHIMERA rTBIAnalysis of high-speed videography (5,000 fps) was usedto assess the biomechanical responses of the head in agroup of 8 mice during CHIMERA rTBI at an impactenergy of 0.5 J (Figure 1; peak kinematic parametersdepicted in Figure 1H). Trajectories of the mouse headin the sagittal plane during peak acceleration followingtwo impacts spaced at 24h are depicted in Figure 1A.Following vertical impact, the head followed a loopedtrajectory in the sagittal plane (Additional file 1: FigureS1, and Additional file 2: Movie S4). The average headtrajectories following two repetitive TBIs in 8 mice werehighly consistent (Figure 1A). The head traveled a peakNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 3 of 18 displacement of 49.6 ± 3.5 mm (mean ± SD, samebelow) in 15.7 ± 2.4 ms (Figure 1B) and exhibited apeak angular deflection of 2.6 ± 0.28 rad in 24.8 ±3.1 ms (Figure 1C). Peak linear velocity was 6.6 ±0.8 m/s at 3.4 ± 1.0 ms (Figure 1D), and peak angularFigure 1 Head kinematics during rTBI. Head kinematic parameters during imthe means for each impact. (A) Head trajectory during the maximum acceleratitime graph following impact. (C) Head deflection is measured as the angle betwand linear head acceleration are depicted in (D) and (E), respectively. (F) and (G(A) are represented as mean ± 95% CI in both X- and Y- direction, respectively. Dof kinematic parameters averaged across all 8 rTBI mice. The coefficient of variatall available recordings.velocity was 305.8 ± 73.7 rad/s at 2.8 ± 1.9 ms followinginitial impactor contact (Figure 1F). The head experi-enced large linear and angular accelerations followingimpact, achieving peak linear acceleration of 385.3 ± 52 gat 1.5 ± 0.3 ms (Figure 1E), whereas the peak angularpacts were assessed in 8 mice subjected to rTBI. Data are represented ason phase in the sagittal plane following impact. (B) Head displacement-een the snout, side marker and the horizontal plane. Linear head velocity) show angular head velocity and angular acceleration, respectively. Data inata in B-G are represented as mean ± 95% CI. (H) Summary of peak valuesion (CV) was calculated as the average of day 1 and day 2 peak values fromacceleration of 253.6 ± 69.0 krad/s2 was observed at 0.8 ±1.1 ms (Figure 1G). As the head was stationary beforeimpact, the change in head velocity (ΔV) equals peak headvelocity and was found to be 6.6 m/s. The energy trans-ferred from the piston to the head was 0.07 J.Using the equal stress/equal velocity approach [25-27]to scale our murine kinematic data to human-equivalentvalues, ΔV was found to be comparable to National Foot-ball League (NFL) values and higher than Olympic boxingvalues, whereas scaled linear and angular velocity andacceleration parameters were lower than NFL values butcomparable to Olympic boxing values (Additional file 3:Table S5).CHIMERA rTBI induces behavioral deficitsLoss of righting reflex (LRR) in animals after TBI is con-sidered analogous to loss of consciousness in humansafter TBI and can be considered as a behavioral indica-tor of injury severity [28]. Mice subjected to CHIMERArTBI showed significantly increased LRR duration com-pared to sham animals (Figure 2A; TBI effect: F(1, 65) =59.61, p < 0.001). LRR duration was consistent betweenthe first and second impacts (Figure 2A). We furtherassessed injury severity using the Neurological SeverityScore (NSS), which is a composite of ten tasks that as-sess motor reflexes, alertness, and physiological behavior[29] (Additional file 4: Table S6). The NSS of injureds oeriNNhowatiahoNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 4 of 18 2 CHIMERA rTBI induces behavioral deficits. (A) Duration of losTBI procedure. Cohort size: Sham, N = 31; rTBI, N = 39. (B) Neurological sevSham (1 h: N = 34, 1d: N = 31, 2d: N = 35, 7d: N = 21); rTBI (1 h, 1d and 2d:rotarod at 1d, 2d, 7d and 14d post-rTBI. Cohort size: Sham (1d and 2d:14d: N = 15). (D) Thigmotaxis was quantified at 1d, 7d and 14d post-rTBI. CoN = 10, 14d: N = 15). Data in A-D were analyzed by repeated measures two-was assessed by the passive avoidance test from 7d to 10d post-rTBI.. (F) SpaData at each time point represent the mean of four trials. Data in E and F (CoANOVA. For all graphs, data are presented as mean ± SEM. For all graphsand # indicates a significant time effect within each group. ***: p < 0.00f righting reflex (LRR) was assessed immediately following the sham orty score (NSS) was assessed at 1h, 1d, 2d, and 7d post-rTBI. Cohort size:= 42, 7d: N = 25). (C) Motor performance was assessed on an accelerating= 35, 7d: N = 21, 14d: N = 15); rTBI (1d and 2d: N = 41, 7d: N = 25,rt size: Sham (1d: N = 23, 7d: N = 16, 14d: N = 15); rTBI (1d: N = 24, 7d:y ANOVA followed by the Holm-Sidak post-hoc test. (E) Working memoryl reference memory was assessed by Barnes maze from 9d to 13d post-rTBI.rt size: N = 15/group) were analyzed by repeated measures two-way, * indicates a significant rTBI effect within a particular time point1. ###: p < 0.001.Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 5 of 18 was significantly higher than sham mice from1h to 7d post-procedure (Figure 2B, TBI effect:F(1, 191) = 44.12, p < 0.001). In injured mice, the NSSscore showed maximum deficits at 1h post-procedure(p < 0.001) followed by steady spontaneous improvementover 1-7d, albeit remaining significantly higher thansham animals at each post-rTBI time point (Figure 2B,p < 0.001). Similarly, rTBI significantly impaired motorperformance from 1-7d post-injury as indicated by re-duced fall latencies on an accelerating rotarod comparedto sham controls (Figure 2C, TBI effect: F(1, 220) =11.99, p < 0.001). Fall latencies showed both time effects(F(4, 220) = 13.70, p < 0.001) and TBI × Time interaction(F(4, 220) = 11.22, p < 0.001). Motor deficits in injuredmice peaked at 1d (p < 0.001) and returned to baselineconditions by 14d post-injury (p = 0.22), whereas shammice did not show any motor deficit (p > 0.82). Injuredmice showed anxiety-like behavior as indicated by sig-nificantly increased thigmotaxis in an open field test(Figure 2D, TBI effect: F(1, 53) = 12.30, p < 0.001). Thethigmotactic behavior of both groups significantly de-clined over time in a similar trend (Figure 2D, Time effect:F(2, 53) = 5.45, p = 0.007; TBI × Time interaction insig-nificant). Open field thigmotaxis was not affected bygross motor activity as no significant differences in totaldistance traveled or time immobile were observed be-tween injured and sham-operated mice (Additional file5: Figure S2). Repeated TBI also induced working mem-ory impairment as indicated by decreased latencies toenter the darkened compartment on the passive avoidancetask (Figure 2E, TBI effect: F(1, 28) = 4.6, p = 0.041). Forall mice, a main effect of Day indicated that mouse behav-ior changed over the training and testing sessions evalu-ated in this study (F(3, 84) = 58.55, p < 0.001). For themain effect of Day, pairwise comparisons indicated thatmice entered the darkened compartment significantlyfaster on Day 7 (p < 0.001) than on Days 8–10 (p > 0.05),signifying that all mice remembered the shock to somedegree (Figure 2E). Injured mice also showed spatial refer-ence memory impairment as indicated by increased laten-cies to locate the escape hole on the Barnes maze(Figure 2F, TBI effect: F(1, 28) = 6.27, p = 0.018). Underour experimental conditions, cognitive performance didnot spontaneously resolve by the end of our testingperiod.CHIMERA rTBI induces widespread persistent diffuseaxonal injurySilver staining was used to assess post-rTBI axonal dam-age at 2, 7, and 14d post-rTBI (Figures 3 and 4). rTBIbrains revealed widespread axonal injury, as indicated byintense punctate and fiber-associated argyrophilic struc-tures in several white matter tracts including the olfac-tory nerve layer of the olfactory bulb, corpus callosum,and optic tracts (Figure 3A and B, Figure 4). Axonalinjury was observed at both coup (corpus callosum) andcontrecoup (optic tract) regions, indicating a diffuse in-jury pattern. High-magnification of the affected areas at100X revealed numerous axonal varicosities (Figure 3C,arrows), which is a characteristic histological feature ofhuman axonal pathology after TBI [30]. Quantitativeanalysis revealed significant silver uptake in the in-jured olfactory nerve layer (TBI effect: F(1, 27) = 16.89,p < 0.0001), which was maximum at 2d (p < 0.001) andreturned to sham levels over 7-14d (Figure 4A). On theother hand, rTBI induced persistent silver stain uptakein the corpus callosum (Figure 4B, TBI effect: F(1, 37) =41.54, p < 0.0001; Time effect insignificant) (Figure 4C).In the optic tract, silver uptake was most intense(Figures 3B and 4C TBI effect: F(1, 36) = 107.4) andthere was a significant time and injury interaction (TBI xTime interaction: F(2, 36) = 11.66), indicating persistentincrease in axonal degeneration in contrecoup regions.CHIMERA rTBI induces widespread microgliosis andincreases proinflammatory cytokine levelsUsing Iba-1 immunohistochemistry, we observed signifi-cantly increased microglial activation throughout severalwhite matter tracts including the olfactory nerve layer,corpus callosum, optic tracts, and brachium of superiorcolliculus of injured brains compared to the sham controlsas assessed using both fractal analysis and microglialdensity (Figures 5 and 6). Quantification of microglialmorphology by fractal analysis revealed that microgliain sham animals displayed highly ramified and exten-sively branched processes that are characteristic of theresting state (Figure 5C, upper row, Figure 6A-D, openbars). By contrast, microglia in the corpus callosum, bra-chium of superior colliculus, and olfactory nerve layer ofinjured animals had predominantly hypertrophic to bushymorphology with primary branches only, whereas those inthe optic tract showed amoeboid morphology characteris-tic of highly activated microglia (Figure 5C, lower row,Figure 6A-D, black bars). Quantitative analysis showedsignificant and persistent microglial activation in the in-jured olfactory nerve layer (TBI effect: F(1, 35) = 13.64,p = 0.0008), optic tract (TBI effect: F(1, 37) = 9.77,p = 0.0034), corpus callosum (TBI effect: F(1, 38) = 29.51,p < 0.0001), and brachium of superior colliculus (TBI ef-fect: F(1, 38) = 24.5, p < 0.0001) as soon as 2d (Figure 6A,B, C and D).In addition to changes in microglial morphology, weobserved significant increases in the number of micro-glia in the same white matter regions including theolfactory nerve layer (Figure 6E, TBI effect: F(1, 16) =21.53, p = 0.0003), optic tract (Figure 6G, TBI effect: F(1,16) = 90.30, p = 0.0001), corpus callosum (Figure 6F, TBIeffect: F(1, 19) = 22.25, p = 0.0002) and brachium ofNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 6 of 18 colliculus (Figure 6H, TBI effect: F(1, 18) =34.85, p < 0.0001), indicating that injury induced prolifer-ation or recruitment of immune cells. In olfactory bulb(Time effect insignificant), corpus callosum (Time effectinsignificant) and optic tract, microglia cell number waspersistently increased from 2d up to 14d (p < 0.05). InFigure 3 CHIMERA rTBI induces diffuse axonal injury. Axonal degeneratiosections showing white matter areas including the olfactory nerve layer,staining indicated by black rectangles. (B) Representative 40X-magnifiedrTBI-induced (lower three rows) animals at the indicated time points. (C) 100XAxonal varicosities are indicated by arrows.the brachium of superior colliculus, a delayed butpersistent increase in the Iba-1 positive cell number wasobserved from 7d to 14d (p < 0.01).In addition to the microglial response, we also mea-sured protein levels of the proinflammatory cytokinesTNFα and IL-1β in half brain homogenates. Proteinn was assessed by silver staining at 2, 7, and 14d post-rTBI. (A) Coronalcorpus callosum, and optic tract with regions of prominent silverimages of the same brain regions in sham-operated (upper row) or-magnified images of the same brain regions in rTBI-induced animals.geDta: N=rT01Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 7 of 18 of TNFα (Figure 7A) and IL-1β (Figure 7B) weresignificantly higher at 2d post-TBI compared to therespective sham levels (p < 0.01).CHIMERA rTBI increases endogenous tau phosphorylationWe next assessed the phosphorylation levels of en-dogenous murine tau using three antibodies directedagainst different phosphorylation sites, namely CP13Figure 4 Quantitative analysis of silver stain images. Silver stained imawhite matter tract area that was stained with silver. Bars indicate mean ± Sin (A) olfactory nerve layer, (B) corpus callosum and (C) optic tract. Datest. Cohort size: olfactory nerve layer: Sham (2d: N = 4, 7d: N = 5, 14d(2d and 7d: N = 5, 14d: N = 6); rTBI (2d: N = 16, 7d: N = 5, and 14 d: N(2d: N = 17, 7d and 14d: N = 5). For all graphs, * indicates a significanttime effect within rTBI group. **: p < 0.01, ***: p < 0.001, ****: p < 0.00(pSer202), RZ3 (pThr231), and PHF1 (pSer396 andpSer404). Total murine tau levels were determined bythe antibody DA9. Simple Western analysis showed sig-nificantly increased phosphorylation of all the probedepitopes in rTBI brain lysates at 6h, 12h, and 2d com-pared to the respective sham brain lysates (Figure 8A-Cand 8G-I, p < 0.01). The change in tau phosphorylationreflected a significant increase in the ratio of phosphor-ylated tau:total tau, but not a change in total tau levels(Figure 8D-F and 8G-I, p < 0.01).DiscussionThe major goal of this study was to develop a simple, reli-able model of murine CHI that replicates fundamentalaspects of human impact TBI through precise delivery ofknown biomechanical inputs. CHIMERA fulfills these cri-teria and offers several key advantages over existing rodentTBI models. CHIMERA is completely nonsurgical and re-quires only isoflurane anesthesia, therefore enabling im-mediate neurological severity assessments using LRR andNSS measures. Being nonsurgical, CHIMERA is ideal forstudies investigating multiple impacts as well as the long-term consequences of impact TBI. These advantages over-come many limitations of surgically-induced TBI models,including longer exposure to multiple anesthetic agents,such as opioid analgesics (buprenorphine) and sedatives(xylazine) that can interfere with rapid neurologicalassessment. Surgical models are also low throughput,require extensive operator training, and have limited suit-ability for studies involving repetitive TBI or long-termTBI outcomes. By contrast, CHIMERA produces injuryusing a simple, reliable, and semi-automated procedures were quantified by calculating the % of region of interest (ROI) in thepercent of ROI showing positive signal in sham and rTBI-induced animalswere analyzed using two-way ANOVA followed by a Tukey post-hoc= 6); rTBI (2d: N = 8, 7d and 14 d: N = 5); corpus callosum: Sham6); optic tract: Sham (2d: N = 5, and 7d: N = 4, 14d: N = 6); rTBIBI effect within a particular time point and # indicates a significant. ###: p < 0.001, ####: p < 0.0001.that requires <10 min per animal to produce defined in-jury (Figure 9). As the biomechanical input parameters arehighly adjustable across impact energy, velocity, and direc-tional parameters, CHIMERA offers a wide dynamic rangeof precisely controllable inputs to reproduce specificconditions that occur in human impact TBI. Impor-tantly, the kinematic analyses facilitated by CHIMERAenable head motion parameters to be integrated withbehavioral and neuropathological outcomes, potentiallyenabling greatly improved translational relevance to hu-man TBI. CHIMERA produces diffuse injury character-ized by activation of inflammatory reactions, axonaldamage, and tau phosphorylation, replicating manyaspects of the neuropathology of human impact TBIwithout overt focal damage. Taken together, these attri-butes make CHIMERA a valuable new model to investi-gate the mechanisms of TBI and for use in preclinicaldrug discovery and development programs.In this study, a 50 g piston was used to deliver an inputimpact with a kinetic input energy of 0.5 J. Calibrationcurves show that the 50 g piston has an energy range of0.01 J to 14.0 J (useful range for murine TBI is 0.1 J to 1 J)in minimum steps of 0.01 J, with highly reproducible per-formance (Figure 9D). Adjusting the piston mass andNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 8 of 18 will allow other biomechanical parameters tobe controlled, so that it may be possible to experimentallymodel the biomechanical conditions observed under dif-ferent types of human impact TBI.High-speed video integrated into CHIMERA enablesmeasurement of mouse head kinematic parameters thatcan be scaled to humans. The most-commonly usedmethod for scaling kinematic parameters between humansand animals is based on the equal stress/equal velocity ap-proach [25-27]. Thus, velocity does not scale and is theFigure 5 CHIMERA rTBI induces widespread microglial activation. Microand 14d post-rTBI. (A) Representative images of Iba-1 stained coronal sectionare indicated by black rectangles. (B) Representative 40X-magnified images obrains (upper row) and activated microglia in injured brains (lower three rowsshowing the morphology of Iba-1-stained resting microglia in sham (upper rosame for human and animal data. We used a scaling factorλ [λ = (mass of human brain/mass of mouse brain)1/3 =13.8] to estimate the human head-equivalent kinematicparameters from our animal data. Although this scalingapproach has been widely used in the study of impactbiomechanics [25-27], and has been applied to a ratCHI model [25], it is important to be cautious in itsextrapolation as the human and rodent brains differ ingeometry, white:grey matter ratio, ventricular volumeand position, and cortical folding. All of these factorsglial activation was assessed using Iba-1 immunohistochemistry at 2, 7,s of olfactory bulb and brain. Areas with prominent microglial activationf the same white matter tract regions showing resting microglia in sham) at the indicated time points. (C) Representative 100X-magnified imagesw) and activated microglia in rTBI (lower row) brains.Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 9 of 18 the “scaling” of rodent data to human to be anapproximation at best. Nevertheless, under our experimen-tal conditions, behavioral and neuropathological changesreliably occurred at lower scaled values of all kinematicFigure 6 Quantitative analysis of microglial response to rTBI. Bar grapmicroglial morphology in (A) olfactory nerve layer, (B) corpus callosum, (C)right column (E-H) show mean ± SD number of Iba-1 positive cells per mmANOVA followed by a Tukey post-hoc test. Numbers inside the bars indicate sparticular time point while # indicates a significant time effect within rTBI gro####: p < 0.0001.parameters (except for impact duration) than those re-ported for NFL concussions [31,32]. Because clinical mTBIcan occur under many different circumstances (e.g., falls,passengers and pedestrians in motor vehicle accidents,hs in the left column (A-D) indicate mean ± SD fractal dimension forbrachium of superior colliculus, and (D) optic tract. Bar graphs in the2 in the same white matter regions. Data were analyzed by two-wayample size. For all graphs, * indicates a significant rTBI effect within aup. *: p < 0.05, **: p < 0.01, ***: p < 0.001, ****: p < 0.0001. ##: p < 0.01,r gresle cNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 10 of 18 sports), an important area for future research willbe to determine how various impact characteristics lead tofunctional and biochemical changes. The precision andflexibility CHIMERA offers with respect to impact parame-ters will help to refine the relationship between impactcharacteristics and physiological outcomes.An additional advantage of CHIMERA is that it over-comes much of the variability observed with most CHImodels. Typical weight-drop models [29,33] have poorcontrol over biomechanical input parameters, includingfriction and air resistance inside the guide tube that maycontribute to variable outcomes. High incidence of skullfractures and limited dynamic range pose additionalchallenges in comparing results using weight-drop TBImodels across different laboratories [24]. Head movementduring many CHI impact models is often at least partiallyrestricted by anchoring the head within a stereotaxicframe or by resting the animal on various types of support.Figure 7 CHIMERA rTBI increases proinflammatory cytokine levels. Bain rTBI brain lysates compared to the levels in the sham brain lysates at theof rTBI vs sham, using multiple t-tests with Bonferroni corrections for multipIn a recent modification, Kane et al. supported mice on apiece of aluminum foil that ruptures upon impact andleads to a 180° rotation of the animal [34]. While thismodification allows unrestricted head movement, it stillincludes possible sources of variability including stretch-ing of the aluminum foil before yielding to the force gen-erated by the weight-drop and less reliable and adjustablepositioning of impact location compared to CHIMERA.Loss of consciousness for <30 min is one of the clin-ical criteria for mTBI [35] and the analogous measure inmice is LRR. In animal TBI models, an LRR of 15–30 minis considered moderate-severe TBI while an LRR of<15 min is considered mild TBI [28]. The average LRRduration after CHIMERA-rTBI was 5.3 min, indicatingmild TBI. Interestingly, a second TBI occurring 24 hafter the first impact did not prolong LRR time. Post-concussion patients may also show balance difficultiesor postural instability up to several days [36-38], aswell as mood changes such as irritability or anxiety [38,39].Though general locomotor activities were not severelyaffected (Additional file 5: Figure S2), CHIMERA-rTBIresulted in deficits in fine motor coordination andneurological performance. Similar changes have alsobeen reported by other groups [40,41]. CHIMERA-rTBIalso increased thigmotaxis in TBI animals, suggestingan anxiety-like behavior [42]. Our model also revealeddeficits in both working and spatial reference memoryas assessed by the passive avoidance test and Barnesmaze, respectively. Intriguingly, recovery of motor per-formance was faster than cognitive performance underthe conditions of our study.Diffuse axonal injury (DAI) is one of the characteristicpathologies of TBI [30,43]. Using silver staining, we ob-served increased argyrophilic fibers and punctate struc-tures in several white matter tracts across the brain,suggesting a diffuse pattern of damaged axons. Axonalvaricosities, a classical feature of DAI in humans, wereraphs represent mean ± SD % fold change in TNFα (A) and IL-1β (B) levelspective time points. For both graphs, ** indicates p< 0.01 in comparisonomparisons (p= 0.05/5 = 0.01).also present [30]. Interestingly, both white matter areasthat were close to (e.g., corpus callosum) and distantfrom (e.g., optic tract and olfactory nerve layer) the im-pact site were affected, suggesting that both coup andcontrecoup injuries are present in our model. Affectedwhite matter areas, including the corpus callosum, optictract, and the olfactory system, have been reported inother CHI models that induce impact at the superiorside of skull [44-47]. Several white matter areas showedincreased argyrophilic staining as well as reactive micro-gliosis following rTBI, suggesting a possible relationshipbetween axonal damage and neuroinflammation, inagreement with previous reports [46,48,49]. Interest-ingly, axonal injury in the optic tract continued to in-crease from 2 to 14d suggesting that ongoing secondaryinjury processes overwhelmed endogenous repair mech-anisms in the time frame examined in this study. More-over, optic tract, a contrecoup injury site, showed themost intense silver uptake, which is in agreement withNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 11 of 18 common clinical observation that contrecoup injuriesare more severe than the coup injuries [50]. On the otherhand, axonal injury was resolved in the olfactory neuronalFigure 8 CHIMERA rTBI increases endogenous tau phosphorylation. Tau(ProteinSimple). The graphs in the left column (A-C) depict fold change in encompared to the sham brains using antibodies CP13 (pSer202 and pThr205, Prespectively. Graphs in the middle column (D-F) depict quantitation of pdigital immunoblots of phosphorylated and corresponding total tau are depicmolecular weight marker at 66 kDa. Data are presented as the mean ± SD foldFor all graphs, *, ** and *** indicate p < 0.01 for the comparison of rTBI vs respmultiple comparison (p = 0.05/5 = 0.01).layer within 7d, suggesting efficient neural repair or activeneuroregeneration in this region. Future studies will bedesigned to assess changes in cognitive functions and thephosphorylation was analyzed using the Simple Western systemdogenous phosphorylated tau levels in rTBI half-brain homogenatesanel A), RZ3 (pThr231, Panel B) and PHF1 (pSer396 and pSer404, Panel C),hosphorylated tau as a proportion of total tau (DA9). Representativeted in the right column (G-I). Arrows on the left of the blots indicatechange in rTBI compared to the respective shams at each time point.ective sham values, using multiple t-tests with Bonferroni correction forre dnk,hoedNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 12 of 18 9 CHIMERA device and mouse head positioning. (A) The pictufollows: 1. head plate, 2. body plate, 3. animal bed, 4. Velcro straps, 5. air tavalve, 9 vertical piston barrel. (B) Close-up view of animal strapped on thebrain. P: impact piston. (D) Air pressure-energy calibration curve was obtaindynamics of axonal injury across several brain regions inour model over longer-term (up to 6 months) post-rTBIfollow up.Human and experimental TBI induce rapid neuroin-flammatory responses as demonstrated by changes incytokine levels (e.g., IL-1β and IL-6) and microglial acti-vation [44,51-55]. Under our injury conditions, rTBI ledto elevated IL-1β and TNF-α levels at 2d after injury,which was accompanied by histological evidence ofmicrogliosis. Because our animal ethics committee re-quired the use of meloxicam for pre-emptive pain control,it is possible that an inflammatory response occurringduring the first few hours after impact [52,54,55] wassuppressed [56]. Iba-1-positive activated microglia wereparticularly evident along white matter tracts throughoutthe brain, whereas grey matter was essentially spared.Microglial activation as assessed by fractal analysis andcell density was significant at 2d and persistent until14d. However, as Iba-1 does not distinguish the sourceof immune cells, the increase in cell number in thisstudy may be due to proliferation of resident immunecells in the brain or recruitment of immune cells fromthe periphery, or both.Hyperphosphorylation of the cytoskeletal protein tauis a pathological event observed in many neurodegene-rative diseases including Alzheimer’s disease [48] andchronic traumatic encephalopathy [49]. In our model, wethe resultant impact energy. The graph depicts three measurements for eachepicts the CHIMERA device. Various parts are labeled with numbers as6. air pressure regulator, 7. digital pressure gauge, 8. two-way solenoidlding platform. (C) Location of impact relative to the mouse head andby driving a 50 g piston at increasing air pressure values and calculatingdemonstrate that endogenous tau hyperphosphorylation isan early and dynamic event after rTBI in wild-type mice,again, in agreement with other models of CHI [57,58]. Itshould, however, be noted that post-TBI changes in phos-phorylation of murine tau does not predict whether hu-man tau will show similar dynamics. Further experimentsusing transgenic human tau mice will be required toinvestigate the influence of rTBI on tau deposition.ConclusionsHere we report a novel, surgery-free CHI model thatfully integrates biomechanical, functional, and neuro-pathological characteristics of TBI. CHIMERA allows pre-cise control over mechanical inputs allowing reproduciblehead kinematics. Our study also shows that CHIMERA-TBI reliably replicates several key behavioral, biochem-ical, and neuropathological characteristics of humanTBI including axonal injury, neuroinflammation, andfunctional deficits. Future studies will be conducted tocharacterize, in more detail, the relationships betweenkinematics and the resulting behavioral and neuro-pathological responses across a variety of impact pa-rameters. The significant advantages CHIMERA offersover comparable rodent TBI models are expected to fa-cilitate the acquisition of preclinical data with improvedrelevance to human TBI, thereby accelerating the paceof successful research to understand the mechanisms ofair pressure value.Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 13 of 18 and to develop effective therapeutic approaches forthis devastating condition.MethodsCHIMERA impactorThe CHIMERA impactor consists of an aluminum framethat supports an animal holding platform above a pneu-matic impactor system (Figure 9). The animal holdingplatform is composed of a fixed head plate that supportsthe animal’s head in a supine position and a body platethat positions and secures the animal’s torso. The headplate has a hole through which the tip of the impactorpiston is projected to contact the animal’s head. A cush-ion made from closed-cell foam surrounds the hole tominimize rebound impact when the animal’s head fallsback upon the head plate. Two perpendicular linesacross the piston hole act as crosshairs for aligning theanimal’s head over the hole. The body plate holds a re-straint system consisting of an animal bed of closed-cellfoam contoured to the shape of the animal’s body andtwo Velcro straps. The animal holding platform is at-tached to the frame by hinges and its angle of inclinationcan be adjusted. In this study, the angle was set to ap-proximately 32° such that the frontal and parietal boneslie flat over the hole in the head plate, thus deliveringimpact to the dorsal cortical region.The pneumatic impactor system includes an accumu-lator air tank, pressure regulator, digital pressure gauge,two-way solenoid valve, and trigger button. The pres-sure regulator and digital pressure gauge allow preciseadjustment of air pressure to 0.1 psi (0.69 kPa), enablingaccurate delivery of piston velocity and impact energy. Im-pact is induced with a 50 g free-floating chrome-coatedsteel piston whose trajectory is constrained to linear mo-tion by a steel barrel. The piston barrel has an array ofholes drilled near the muzzle end to vent air and equalizethe pressure as the piston moves past them towards theimpact site. The piston is accelerated by a controlled pulseof compressed air along the length of the barrel until itclears the venting holes.The CHIMERA impactor was calibrated by measuringthe exit velocity of the piston at various air pressures(0.5, 1, 1.5, 2, 3, 5, 7, and 10 psi) to determine the rela-tionship between air pressure and piston velocity. Threemeasurements were taken at each pressure value. Eachimpact event was recorded by a high-speed video cameraat 10,000 fps and tracked by video motion analysis soft-ware (TEMA Motion, Image Systems AB, Sweden). A2nd order polynomial curve was used to fit the data.The r2 value was 0.9996 (Figure 9D). Using this curve,the desired impact velocity or energy can be independ-ently interpolated. By choosing the appropriate air pres-sure, impacts of input energy ranging from 0.01 J to 1 Jcan be precisely generated.CHIMERA TBI procedureAll animal procedures were approved by the Universityof British Columbia Committee on Animal Care (proto-col # A11-0225) and were carried in strict accordancewith the Canadian Council on Animal Care guidelines.Male C57Bl/6 mice (mean ± SD body weight 33.9 ±4.6 g) at 4 months of age were housed with a reversed12h light-12h dark cycle for at least 10 days before TBI.Animals were anaesthetized with isoflurane (induction:4.5%, maintenance: 2.5-3%) in oxygen (0.9 L/min). Lu-bricating eye ointment was applied to prevent cornealdrying. Meloxicam (1 mg/kg) and saline (1 mL/100 gbody weight) were administered by subcutaneous injec-tions for pain control and hydration, respectively. Ani-mals were placed supine in the holding bed such thatthe top of the animal’s head lay flat over a hole in thehead plate, aligned using crosshairs such that the pistonstrikes the vertex of the head covering a 5 mm area sur-rounding the bregma (Figure 9B and C). Impact was in-duced by pressing a trigger button that simultaneouslyfires the piston and when connected, activates a high-speed camera to record the resulting head trajectory.Isoflurane delivery was immediately stopped and the ani-mal was continuously monitored until fully ambulatory.Total duration of isoflurane exposure was ~ 4–8 min.Twenty-four hours after the first impact, a second iden-tical impact was delivered. Sham animals underwent allof these procedures, except for the impact. Approxi-mately 3% of animals did not regain consciousness for >45 min or displayed severe motor dysfunction after TBI,and were thus euthanized.High-speed videography and kinematic analysisFor kinematic analysis, an independent cohort of 8 micewas subjected to rTBI and impact events were recordedat 5,000 frames per second using a high-speed videocamera (Q-PRI, AOS Technologies, Switzerland). Headmotion was tracked using two markers, one being non-toxic paint applied on lateral side of head to mark thecheek area (Additional file 1: Figure S1, yellow arrow inthe first image). Because the skin is loose over the bonyskull, we also marked the position of the maxilla bywrapping dental floss positioned just caudal to theupper incisors around the animal’s snout (Additionalfile 1: Figure S1, red arrow in the first image). Videoswere analyzed using ProAnalyst motion analysis soft-ware (v, Xcitex Inc., Woburn, MA). The X andY coordinates of the position of each marker weretracked on a frame-by-frame basis and were processedwith a 400-Hz low-pass Butterworth filter to mitigatethe noise in the recorded measurements. Velocities andaccelerations were determined by discrete differenti-ation of the position data. Resultant linear velocity andacceleration were calculated as the magnitude of theirNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 14 of 18 X and Y components. Linear kinematic pa-rameters were assessed from trajectories obtained bytracking the paint mark. Angular rotation of the headduring TBI was determined by the angle of the linejoining the dental floss and the paint mark with thehorizon. After video capture, the dental floss was re-moved. Energy transferred from the piston to the headwas determined using the equation KE = 0.5 × Me ×ΔV2, where Me is the effective mass and is approximatedby head mass (3.4 g) and ΔV is a change in head velocity.A scaling factor λ [λ = (mass of human brain/mass ofmouse brain)1/3 = 13.8] was used to estimate the humanhead-equivalent kinematic parameters from the animaldata.Behavioral analysesLRR was calculated as the time interval from isofluranediscontinuation to the first sign of righting after eachimpact. Neurological impairment was assessed using theNSS [29] determined at 1h and at 1, 2, and 7d followingthe second TBI. NSS is a composite of ten different tasksthat assess motor function, alertness, and physiologicalbehavior (Additional file 4: Table S6). One point isawarded for the lack of a tested reflex or for the inabilityto perform the tasks, and no point for succeeding thetask. A maximal NSS of 10 points thus indicates severeneurological dysfunction, with failure of all tasks. Motorperformance was evaluated at 1, 2, 7, and 14d after thesecond TBI using an accelerating rotarod as previouslydescribed [46]. Open field activity was assessed at 1, 7,and 14d after the second TBI using a Plexiglas box(14” × 24” × 14”). The floor of the box was virtuallydivided into 60 equal squares using an overhead digitalcamera and video tracking software (ANY-maze, v.4.99, Stoelting Co, Wood Dale, IL). The field wasfurther subdivided into a peripheral zone along thewalls of the open field consisting of 28 squares thatsurrounded a central zone consisting of 32 squares.The animal was placed in the center of the box andspontaneous activity was recorded for 10 min, includingquantification of the total distance traveled and immo-bile time. The thigmotaxis index (TI) was calculated as:TI = (TP-TC)/(TP + TC) where TP and TC represent thetime spent in the peripheral and central zones, respect-ively. Working and spatial reference memories wereassessed from 7d to 13d after the second TBI using thepassive avoidance task (7-10d) and the Barnes maze(8-13d), respectively. Passive avoidance testing wasconducted in a device that consisted of two adjoiningcompartments, one illuminated (20.3 × 15.9 × 21.3 cm)and one darkened (20.3 × 15.9 × 21.3 cm), divided bya guillotine-style door (Med Associates Inc., St. Albans,VT). The floor of the compartments consisted ofsteel rods capable of delivering an electric foot-shock.The electric shock was delivered by a programmableanimal shocker (Med Associates Inc.). Each session con-sisted of placing mice into the illuminated compartmentand using a timer to record the latency of the mice to crossinto the darkened compartment. On 7d after the secondTBI (training), mice received an electric foot-shock(0.3 mA, 2 s) as soon as they crossed from the illuminatedinto the darkened compartment. Following foot-shock,mice were removed from the apparatus and returned totheir home cage. On 8d to 10d after the second TBI micewere tested for memory retention. The latency for themice to cross into the darkened compartment wasrecorded. No shock was delivered during testing. Micethat did not cross over into the darkened compartmentwere allowed to remain in the illuminated compart-ment for the full 5 min and assigned a latency of 300 s.Barnes maze testing was conducted on a grey circularplatform (91 cm diameter) with 20 circular holes (5 cmdiameter; Stoelting Co) located in a 3.0 × 3.6 m room.An escape box was positioned beneath one of the holes.Extra-maze visual cues consisted of black and whiteimages (cross, lightning bolt, smiley face) printed onglossy white paper and placed on the walls surroundingthe maze. Other visual cues included a cart with alaptop and the experimenter. A digital camera wasplaced 4 m above the center of the maze to record trialsfor the ANY-maze tracking system. For motivationalpurposes, mice were food restricted to 1 g of foodper day from 8-13d after the second TBI and main-tained at 90% of free feeding body weight. During test-ing, mice were exposed to aversive stimuli in the formof two lights (100 W) positioned at either side of themaze and a buzzer (2.9 kHz, 65 dB) that hung 10 cmabove the maze. On 8d after the second TBI, mice com-pleted one 5-min habituation trial to become familiarwith the maze environment and to practice descendinginto the escape box. On 9d to 13d after the secondTBI, mice were tested for memory retention. Mice weregiven four trials per day (15-min inter-trial interval)for five days. For each trial, mice were placed in ablack plastic start tube (7 cm diameter, 12 cm height)on the center of the maze. After 10 s, the start tube wasraised and the buzzer was turned on to start thetrial. Mice that were unable to locate the escape hole in90 s were gently guided to it. Mice remained in theescape box for 10 s before being returned to theirhome cage.Tissue collectionFor histological analyses, mice were anesthetized with anintraperitoneal injection of 150 mg/kg ketamine (Zoetis)and 20 mg/kg xylazine (Bayer) at 2, 7, or 14d after thesecond TBI, and brains were collected from perfused an-imals as described [46], except that 4% paraformaldehydelated at Ser396 and Ser404 (1:25), CP13 is directed againstNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 15 of 18 than neutral buffered formalin was used to post-fixhemisected brain tissue for histology. For biochemicalanalyses, brains were harvested as above at 6 h, 12 h, 2d,7d, and 14d post-rTBI, longitudinally hemisected and rap-idly frozen over dry ice and stored at −80°C until analysis.Microglial activation and silver stainingSilver staining and Iba-1 histology was performed asdescribed [46]. Microglial morphology was quantifiedusing fractal analysis [59] using ImageJ (NIH). Threeto four microglia were randomly chosen from 40X-magnified Iba-1 stained images of olfactory nerve layer,corpus callosum, optic tract, and brachium of superiorcolliculus, converted to 8-bit images, and thresholded.Thresholded images were converted to outline imagesand analyzed using the FracLac plugin (Karperien, A.,FracLac for ImageJ. 1999–2013.). The boxcounting method was used and the mean fractal dimen-sion was analyzed. The number of microglia in each whitematter regions was quantified by scanning the entire areausing a Olympus BX61 microscope at 10X magnificationwith a ProScan motorized XY stage (Prior Scientific Inc,Rockland, MA). The component images were stitched to-gether into a montage using ImagePro Plus image analysissoftware (Media Cybernetics Inc., Rockville, MD). Thenumber of Iba1-positive cells were manually counted in theentire white matter track region of interest (ROI). The areaof ROI was measured by ImageJ as pixels and scaled tomm2. Cell density was finally expressed as number of Iba-1positive cells per mm2. Silver staining intensity was quanti-fied using ImageJ (version 1.48, NIH) on 40X-magnifiedimages of olfactory nerve layer, corpus callosum, and optictract. The images were thresholded and the ROI wasmanually selected. The ratio of area of positive signal inROI to total ROI area was reported as percent positive.Biochemical analysesTissue processingFor protein determination, half-brains were homogenizedin RIPA lysis buffer as described [46].Cytokine ELISAEndogenous TNFα and IL-1β protein levels in the half-brain homogenates were quantified by commercial ELISAkits (BD Biosciences OptEIA 559603 and 555268, respect-ively) following the manufacturer’s instructions.Quantitative assessment of phosphorylated and total Tauby Simple Western analysisPhosphorylated and total tau were assessed using an auto-mated capillary electrophoresis-sized-based [60,61] SimpleWestern system using the Wes machine (ProteinSimple,San Jose, CA). Simple Western is a gel-free, blot-free,tau protein phosphorylated at Ser202 and Thr404 (1:25),and DA9 is directed against phosphorylation-independent(total) tau (1:5000). GAPDH (clone 6C5, 1:5000, Chemi-con) was used as a loading control. Levels of phosphory-lated and total tau were normalized to GAPDH. Levels ofphosphorylated tau were expressed as fold differencecompared to sham controls at the respective time points.The rTBI protocol and post-rTBI end points aresummarized in Additional file 6: Figure S3.Statistical analysesThe head kinematics data and graphs are presented asmean ± 95% CI. Behavioral data and graphs are presentedas mean ± SEM. All other data and graphs are presented asmean ± SD unless otherwise specified. NSS, LRR, thigmo-taxis, and rotarod data were analyzed using repeated mea-sures two-way ANOVA followed by the Holm-Sidak post-hoc test, as animals were tested repeatedly until sacrifice.Passive avoidance and Barnes maze data were analyzed byrepeated measures two-way ANOVA. Iba-1 and silver stain-ing data were analyzed by two-way ANOVA followed byTukey’s post-hoc test. For all the above statistical analyses,a p value of <0.05 was considered significant. Tau phos-phorylation and cytokine protein expression at each post-rTBI time point was compared to the respective shamvalues by t test followed by Bonferroni correction of mul-tiple comparison, with p value set to <0.01 (5 comparisons),for detecting statistical significance. Statistical analyses ofbehavioral data were performed using SigmaPlot (version12.5, Systat Software Inc.). Statistical analyses for the rest ofcapillary-based, automated protein immunodetectionsystem that automates all the steps following samplepreparation including sample loading, size-based pro-tein separation, immunoprobing, washing, detection,and data analysis. All procedures were performed withmanufacturer’s reagents according to the user manual.Briefly, 5 μL of RIPA lysate (2 μg of protein) was mixedwith 1.2 μL of 5× fluorescent master mix and heated at95°C for 5 min. The samples, blocking reagent, washbuffer, primary antibodies, secondary antibodies, andchemiluminescent substrate were dispensed into desig-nated wells in the manufacturer-provided microplate.Following plate loading, separation and immunodetectionwere performed automatically using default settings. Datawere analyzed with Compass software (ProteinSimple).Samples were immunodetected using following monoclo-nal antibodies (all kind gifts from Dr. Peter Davies, AlbertEinstein College of Medicine, Manhasset, NY, USA): RZ3is directed against tau protein phosphorylated at Thr231(1:25), PHF1 is directed against tau protein phosphory-the data were performed using GraphPad Prism (version6.04, GraphPad Software Inc).Nat Rev Neurol 2013, 9:222–230.Namjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 16 of 18 filesAdditional file 1: Figure S1. CHIMERA allows unrestricted headmotion during TBI. Before impact, the mouse head was freely supportedon a foam pad in the supine position. Velcro straps were applied to thetorso. Impact from the piston deflects the head, which then subsequentlyreturns to its original position on the foam pad. The images were takenat 5,000 fps, at an angle perpendicular to the direction of impact andalong the mouse sagittal plane. Each image shown was 12 ms apart.Head movement was tracked using two markers: a dental floss (redarrow in the first image) wrapped around the maxilla and a non-toxicpaint (yellow arrow in the first image) applied at the lateral size of thehead.Additional file 2: Movie S4. Mouse head motion following impactusing CHIMERA. The impact event was recorded at 5,000 fps. The videoshows movement of the mouse head following impact by thepneumatically-driven piston.Additional file 3: Table S5. Comparison of kinematic parametersbetween rodent TBI models and human TBI [25,31,32,57,62-69].Additional file 4: Table S6. Neurological severity score tasks [29].Additional file 5: Figure S2. CHIMERA rTBI does not affect generalmobility. General mobility was tested by the open field test at 1, 7 and14d post-injury. No significant differences were observed between shamand rTBI mice in total distance travelled (A), number of lines crossed (B),or time spent immobile (C). Data are presented as the mean ± SEM andanalyzed by repeated measures two-way ANOVA followed by Holm-Sidakpost-hoc test.Additional file 6: Figure S3. Experimental Plan. The figure indicatesthe timeline for rTBI/sham procedure and behavioral, biochemical andhistological end points at various post-rTBI time points used in this study.BM: Barnes maze, Iba-1: Iba-1 immunohistochemistry, NSS: neurologicalseverity score, OF: open field behavior, PA: passive avoidance, RR: rotarod,SS: silver stain.AbbreviationsCCI: Controlled-Cortical impact; CHI: Closed-Head injury; CHIMERA: Closed-HeadImpact Model of Engineered Rotational Acceleration; DAI: Diffuse axonal injury;FP: Fluid percussion; LRR: Loss of righting reflex; mTBI: Mild traumatic braininjury; MVA: Motor vehicle accident; NFL: National football league;NSS: Neurological severity score; TBI: Traumatic brain injury; rTBI: Repeatedtraumatic brain injury.Competing interestsThe authors declare that they have no competing interests.Authors’ contributionsThe CHIMERA device was conceived and designed by DRN, WC, PAC, andCLW and constructed and tested by KAM. DRN and WC carried out TBIprocedures, collected head kinematics and behavioral data, performedhistology and analyzed majority of the data. KMM carried out cognitivetesting and analyzed cognitive testing data. MC provided technical supportfor TBI procedures and histology. Biochemistry samples were processed andtau immunoblotting was carried out by AW. Cytokine ELISA was carried outby JF. JR and AH provided support for histology. The manuscript was writtenby DRN, WC, and CLW and critically reviewed by PAC. All authors read andapproved the manuscript.AcknowledgementsThis work was supported by an operating grant from the Canadian Institutesof Health Research to CLW (MOP 123461). DRN was supported by anAlzheimer Society Research Program (ASRP) Doctoral Award (AlzheimerSociety of Canada, WHC is supported by an ASRP Doctoral Award andComissao Technica de Atribuicao de Bolsas para Estudos Pos-GraduadosMacao. The funders had no role in study design, data collection and analysis,decision to publish, or preparation of the manuscript. We are indebted toDr. Peter Davies (Albert Einstein College of Medicine) for the kind gifts of tauantibodies.15. Smith DH, Johnson VE, Stewart W: Chronic neuropathologies of single andrepetitive TBI: substrates of dementia? Nat Rev Neurol 2013, 9:211–221.16. Gavett BE, Stern RA, McKee AC: Chronic traumatic encephalopathy: apotential late effect of sport-related concussive and subconcussive headtrauma. Clin Sports Med 2011, 30:179–188. xi.17. Blumbergs PC: Pathology. In Head Injury - Pathophysiology and Managementof Severe Closed Head Injury. Edited by Reilly P, Bullock R. London: Chapmanand Hall; 1997:39–70.18. McIntosh TK, Smith DH, Meaney DF, Kotapka MJ, Gennarelli TA, Graham DI:Neuropathological sequelae of traumatic brain injury: relationship toneurochemical and biomechanical mechanisms. Lab Invest 1996,74:315–342.19. Davis AE: Mechanisms of traumatic brain injury: biomechanical, structuralAuthor details1Department of Pathology and Laboratory Medicine, The University of BritishColumbia, Vancouver, BC, Canada. 2Graduate Program in Neuroscience, TheUniversity of British Columbia, Vancouver, BC, Canada. 3Departments ofMechanical Engineering and Orthopaedics, The University of BritishColumbia, Vancouver, BC, Canada. 4International Collaboration on RepairDiscoveries, The University of British Columbia, Vancouver, BC, Canada.5Djavad Mowafaghian Centre for Brain Health, The University of BritishColumbia, Vancouver, BC, Canada.Received: 23 June 2014 Accepted: 20 November 2014Published: 1 December 2014References1. Langlois JA, Rutland-Brown W, Wald MM: The epidemiology and impact oftraumatic brain injury: a brief overview. J Head Trauma Rehabil 2006,21:375–378.2. 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Peng Y, Yang J, Deck C, Otte D, Willinger R: Development of head injuryrisk functions based on real-world accident reconstruction. Int JCrashworthiness 2013, 19:105–114.doi:10.1186/1750-1326-9-55Cite this article as: Namjoshi et al.: Merging pathology with biomechanicsusing CHIMERA (Closed-Head Impact Model of Engineered RotationalAcceleration): a novel, surgery-free model of traumatic brain injury.Molecular Neurodegeneration 2014 9:55.Submit your next manuscript to BioMed Centraland take full advantage of: • Convenient online submission• Thorough peer review• No space constraints or color figure charges• Immediate publication on acceptance• Inclusion in PubMed, CAS, Scopus and Google Scholar• Research which is freely available for redistributionNamjoshi et al. Molecular Neurodegeneration 2014, 9:55 Page 18 of 18 your manuscript at


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